MIFETM System Overview

This page outlines the theory and has information on the MIFE system, the CHART and MIFEFLUX software.

Microelectrode ion flux measurement - principles and basic theory

Chemicals in solution move under the influence of chemical forces of diffusion directed towards lower concentration regions. Ions, which are charged, also experience electrical forces if an electric field is present as well. The movement of an ion in solution can be described in terms of these chemical and electrical driving forces and other parameters of the ion and solution. It can be shown that the net flux of an ion, typically measured in units of nmol m-2 s-1, may be found from a measurement of the change in voltage of an ion selective microelectrode that is moved through a small known distance in the solution. This technique allows non-invasive measurement of net ion fluxes through a tissue boundary with resolution of 10 seconds in time and 20 micrometer in position. A suitable microscope is used to observe the microelectrodes and the tissue near which they are moved.

In the diagram a microelectrode, whose tip is filled with the liquid ion exchanger LIX, is initially at a distance x from the tissue into which ions are moving with a net flux J. diagramIt is assumed that there is no bulk solution flow, so that ionic movement in the solution (regardless of the membrane transport processes) is solely by diffusion under the influence of electric and chemical forces in solution. It is also assumed that the measurement is close to the surface that the ionic movement is normal to the surface. The electrochemical potential in the solution at the distance x is µ (joules mol-1). Because the LIX allows free passage of the ion in question (but no others), the electrochemical potential of the ion inside the electrode is also µ. The chemical component of µ inside the electrode is fixed by the concentration of the filling solution; the electrical component is given by zFV , where V (volts) is measured by an electrometer connected via suitable half cells to the electrode solution and to a reference electrode some distance away in the bath solution. The ion's valence is z and F is the Faraday number. The microelectrode is now moved slowly (not to disturb the solution) away through a small distance dx. (It is shown offset in the diagram for clarity only.) At this new position in solution the electrochemical potential is µ + dµ. It is the same inside the electrode, but only the electrical component there has changed, and the measured voltage is now V + dV.

Basic electrochemical theory shows that the net ionic flux J is given in terms of the ion concentration c (mol m-3), the mobility of the ion u (speed per unit force, m s-1 per newton mol-1), and the force per mole which is the electrochemical potential gradient (dµ/dx). Thus J = c u (dµ/dx). But dµ is the same inside the electrode as in the bath solution, and in the electrode dµ = zFdV because the concentration inside is fixed. Hence the flux may be written J = c u z F(dV/dx). The concentration is known, or is adequately measured by the value of V when the electrode has been calibrated in standard solutions. For the ion, u and z are known constants, although for multi-valent ions u depends on z. The electrometer measures dV as the electrode is moved through the chosen distance dx.

The theory can also be expressed in terms of the the diffusion coefficient D for the ion instead of the (related) mobility u. The theory must also be modified to apply to spherical or cylindrical tissues. These alternatives, and the many practical qualifications and limitations, are discussed in the literature, and particularly in the definitive review in the January 2001 Plant, Cell & Environment.
There appear to be two systems designed to implement this technique. Both systems have a sampling rate > 10 Hz and both minimise noise by digital averaging over longer time periods. Thus it would appear that both approaches have the same ultimate limitation on their sensitivity which is set by the thermal electronic noise in the high resistance of the ion selective liquid ion exchanger (LIX) in the micropipette tip. This theoretical limit is discussed in Ryan et al. (1990). That and other references to work using the technique are available.

The MIFE system described below, and in more detail in the review, was developed in Tasmania. The other, which is based on the Vibrating Probe, was developed at Woods Hole in the USA and has been used by Jaffe and various co-workers (eg Kuhtreiber & Jaffe, 1990). [to top of page]

The MIFETM System for ion flux measurement

AmplifierThe MIFE system uses a stepper motor-driven micromanipulator to move four ion selective microelectrodes that measure the electrochemical potential of the ions at two positions in solution close to a tissue surface. Custom-built electronic amplifiers are provided. The CHART program is used to control the data acquisition. From the electrochemical potential difference so measured, the net flux of the ion in solution is calculated using the MIFEFLUX program.

The MIFE system components developed in Tasmania are available for purchase on a commercial basis. These are indicated in bold in the following summary.

System component summary

Electrophysiology lab with bench and Faraday cage.
Microelectrode preparation and filling facilities.
Microscope with vertical or horizontal optic axis as required for the application.
Hydraulic micromanipulator or piezoelectric translator with its amplifier and power supply.
Stepper motors and their drives and power supplies (for the hydraulic manipulator).
Multiple electrode mount.
Manual micromanipulator for adjusting electrode or chamber position.
Computer: PC 286 at least, with DOS, at least 250MB HDD and archiving facility. DAS08 data acquisition card. MIFE electronics: main amplifier and two 4-channel preamplifiers.
General purpose software including spreadsheet and (if needed) Borland Pascal.

Many other things are needed for setting up a MIFE laboratory to measure ion fluxes. We have provided an outline of these requirements with estimates of costs and sources of supply.

The CHART program to record data

CHART is the software package, running under DOS, designed to control data acquisition by the MIFE hardware system. This software allows automated and interactive real-time control of the amplifier configuration and the micromanipulator while the data is being collected and written to disk. The system configuration is recorded together with the data, and all modifications during data acquisition are recorded in a log file which can also include annotations typed during the experiment. Up to 8 channels of data are displayed on the screen as if on a chart recorder. Any region of the “chart” can be inspected (and expanded or contracted) at will without interrupting the measurements. The software allows subsequent recall and display of any run. It also allows the export of the raw or averaged data in the form of an ASCII file for import into a spreadsheet or other program. The system will also function as an excellent electrometer/recorder with a 10 Hz bandwidth for microelectrode studies of membrane potential or for any other data acquisition. [to top of page]

The MIFEFLUX program to calculate ion fluxes

MIFEFLUX was developed to implement the flux calculations according to the published procedures and to provide the necessary software for purchasers of the MIFE amplifiers and controller. It takes output files from CHART and produces convenient ASCII text files for spreadsheet importing. Users who wish to modify the analysis routines will require their own copy of Turbo Pascal or Borland Pascal to edit and compile the Pascal source code which will be provided to MIFE system purchasers. Commercial data manipulating and display software will be needed to graph the calculated fluxes or other data. 

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